%0 Journal Article
%A Jiang, Rui
%A Vandal, Steven
%A Park, SooHyun
%A Majd, Sheereen
%A Tüzel, Erkan
%A Hancock, William O.
%T Slow Microtubule Binding Kinetics of Membrane-bound Kinesin Predicts High Motor Copy Numbers on Intracellular Cargo
%D 2019
%R 10.1101/627174
%J bioRxiv
%P 627174
%X Bidirectional vesicle transport along microtubules is necessary for cell viability and function, particularly in neurons. When multiple motors are attached to a vesicle, the distance a vesicle travels before dissociating is determined by the race between detachment of the bound motors and attachment of the unbound motors. Motor detachment rates (koff) can be measured via single-molecule experiments, but motor reattachment rates (kon) are generally unknown, as they involve diffusion through the bilayer, geometrical considerations of the motor tether length, and the intrinsic microtubule binding rate of the motor. To understand motor attachment dynamics during vesicle transport, we quantified the microtubule accumulation rate of fluorescently-labeled kinesin-1 motors in a 2D system where motors were linked to a supported lipid bilayer. From the first-order accumulation rate at varying motor densities, we extrapolated a koff that matched single-molecule measurements, and measured a two-dimensional kon, which was considerably slower than expected based on the motor tether length. By incorporating cholesterol to reduce membrane diffusivity, we demonstrate that this slow kon is not limited by the motor diffusion rate. Using a theoretical model to interpret our data and a computational model to simulate our experiments, we conclude that the slow kon arises from the fact that motors bind to microtubules only from a small zone, measuring roughly the area of one tubulin dimer, close to the microtubule. The slow kinesin-1 on-rate predicts that long-range transport of membrane-bound vesicles in vivo requires tens to hundreds of motors per vesicle.Significance Statement Long-distance transport of membrane-coated vesicles involves coordination of multiple motors such that at least one motor is bound to the microtubule at all times. Microtubule attachment of a membrane-bound motor comprises two steps – diffusing through the lipid bilayer to a binding zone near the microtubule, followed by binding. Using a 2D supported lipid bilayer system, we show that membrane diffusion is not the limiting factor for motor attachment. Instead, motor attachment is intrinsically slow because motors must be very close to the microtubule to bind. This suggests that higher motor densities than previously anticipated are required for long-range vesicle transport, and that altering membrane composition to change motor diffusion is not an effective mechanism of bidirectional transport regulation.
%U https://www.biorxiv.org/content/biorxiv/early/2019/05/03/627174.full.pdf